Cultivation of micro-organisms on a feedstock consisting at least in part of a straight chain hydrocarbon



United States Patent 3,268,419 CULTIVATION OF MICRO-ORGAIIHSMS ON AFEEDSTOCK CONSISTING AT LEAST IN PART 0F A STRAIGHT CHAIN HYDRO- CARBONAlfred Champagnat and Bernard Laine, Paris, France, assiguors to TheBritish Petroleum Company Limited, London, England, a Britishjoint-stock corporation No Drawing. Filed July 24, 1963, Ser. No.297,213 Claims priority, application France, Aug. 3, 1962, 906,076 18Claims. (Cl. 195-82) The invention relates to a process for theproduction on hydrocarbons of edible micro-organisms in purified form,for example, yeast, bacteria of the orders of Pseudomonadales, Eubacteriales, Actinomycetales and Penicillium expansum.

The cultivation of yeasts on hydrocarbons, and in particular onpetroleum hydrocarbons, as the carbonaceous substratum, generally givesyeasts which after washing and drying possess a peculiar taste which issharp and rancid and which may interfere with their use in foods forhuman consumption. The traditional yeasts, cultivated on molasses,residuary liquors or bisulphite liquors, also have a peculiar taste,different from that of yeasts cultivated on hydrocarbons, and certainprocesses have been proposed for attenuating this tastenotably toattenuate their bitterness, but none of these has succeeded completely.

It has now been found that yeasts cultivated with hydrocarbons as thesole source of carbon have a distinctly higher lipid content than yeastscultivated on the traditional substrate: molasses, residuary liquors,bisulphite liquors. Lipids consist essentially of fatty acids, esters(with greater or lesser degrees of oxidation) including fats andsterols. It has now been found that extraction of these lipids, whollyor in part, leads to a substantial reduction in this characteristictaste of yeasts cultivated on hydrocarbons or to the elimination of thistaste.

It is an object of this invention to provide an improved process for theproduction of micro-organisms. It is a further object to provide aprocess for the production of a yeast. It is a further object to providea process for the removal of straight chain hydrocarbons, wholly or inpart, from mixtures of said hydrocarbons with other hydrocarbons. Otherobjects will appear hereinafter.

According to one aspect of this invention there is provided a processwhich comprises cultivating a microorganism in the presence of afeedstock consisting wholly or in part of straight chain hydrocarbons,separating from the product a fraction comprising the micro-organism andextracting said fraction by means of a solvent.

Solvents which may be employed include ethyl alcohol, isopropanol, lighthydrocarbons, including benzene and light platformate fractions, ethylether, acetone, chlorinated solvents and liquefied petroleum gases, suchas butane and propane.

Within the term micro-organism used herein we'include mixtures ofmicro-organisms.

Micro-organisms which are cultivated as herein described may by yeasts,moulds or bacteria.

Preferably when a yeast is employed this is of the familyCryptococcaceae and particularly of the sub-family Cryptococcoideae;however, if desired there may be used, for example, ascosporogeneousyeasts of the sub-family Saccharomycoideae. Preferred genera of theCryptococcoideae sub-family are Torulopsis (also known as Torula),Candida and Mycoderma. Preferred strains of yeast are as follows. Inparticular it is preferred to use the specific stock of indicatedreference number;

these reference numbers refer to CBS stock held by the Centraal Bureauvor Schimmelculture, Baarn, Holland and to INRA stock held by theInstitut National de la Recherche Agronomique, Paris, France.

Candida lipolytica CBS 599 Candida pnlcherrima CBS 610 Candida utilisCBS 890 Candida utilis., var. major CBS 841 Candida ti-opicalis CBS 2317Candida arbor-ea Torulopsis collz'culosa! CBS 133 Hansenula anomala CBSOidinm laciis Neurospora sitophila Mycoderma cancoillote INRA; STV 11Bacillus amylobacter Pseudomonas natriegens Arthrobacter sp. Micrococcussp. Corynebacterium sp. Pseudomonas .syringae Xanthomonas begoniaeFlavobacterium devorans Acetobdcter sp. Actinomyces sp.

For the growth of the micro-organism it will be necessary to provide, inaddition to the feedstock, an aqueous nutrient medium and a supply ofoxygen, preferably in the form of air.

A suitable nutrient medium for yeasts (and moulds) has the composition:

Grams Diammonium phosphate 2 Potassium chloride 1.15 Magnesium sulphate,7H O 0.65 Zinc sulphate 0.17 Manganese sulphate, 1H O 0.045 Ferroussulphate, 7H O 0.068 Tap water 200 Yeast extract 0.025

Distilled water (to make up to 1000 mls.).

A typical nutrient medium for the growth of Nocardia,

a genus in the Actinomycetales order, has the following composition:

Grams Ammonium sulphate 1 Magnesium sulphate 0.20 Ferrous sulphate, 7H O0.005 Manganese sulphate, 1H O 0.002 Monopotassium phosphate 2 Disodiumphosphate 3 Calcium chloride 0.1 Sodium carbonate 0.1 Yeast extract0.008

Distilled water (to make up to 1000 mls.)

Made up to 1000 mls. with distilled water.

Another suitable nutrient medium for the growth of bacteria has thecomposition:

Grams NH Cl 0.5 NaCl 4 Na HPO 0.5 KH PO 0.5

Water to make up to 1000 cc.

Micro-organisms, and in particular yeasts, when first cultivated withthe use of hydrocarbon fractions as feedstock sometimes grow withdifficulty and it is sometimes necessary to use an inoculum of amicro-organism which has previously been adapted for growth on thehydrocarbon fraction which it is intended to use. Furthermore themicro-organism although cultivated in the presence of an aqueous mineralmedium containing the appropriate nutrient elements may grow withdifficulty, because the hydrocarbon fraction does not contain the growthfactors which exist in carbohydrate feedstocks, unless these growthfactors are added.

In batch operation, the micro-organism will usually grow initially at alow rate of increase in cellular density. (This period of growth isreferred to as the lag phase) Subsequently the rate of growth willincrease to a higher rate of growth; the period at the higher rate ofgrowth is referred to as the exponential phase and subsequently againthe cellular density will become constant (the statitonary phase).

A supply of the micro-organism for starting the next batch willpreferably be removed before the termination of the exponential phase.

The growth operation will usually be discontinued before the stationaryphase.

At this stage, the micro-organism will usually be separated f'rom thebulk of the aqueous nutrient medium and from the bulk of the unusedfeedstock fraction.

The growth of the micro-organism used is favoured by the addition to theculture medium of a very small proportion of extract of yeast (anindustrial product rich in vitamins of group B obtained by thehydrolysis of a yeast) or more generally of vitamins of group B and/ orbiotin. This quantity is preferably of the order of 25 parts per millionwith reference to the aqueous fermentation medium. It can be higher orlower according to the conditions chosen for the growth.

The growth of the microorganism takes place at the expense of thefeedstock fraction with the intermediate production of bodies having anacid function, principally fatty acids, in such manner that the pH ofthe aqueous mineral medium progressively diminishes. If one does notcorrect it the growth is fairly rapidly arrested and the concentrationof the micro-organism in the medium, or cellular density, no longerincreasees so that there is reached a so-called stationary phase.

Preferably therefore the aqueous nutrient medium is maintained at adesired pH by the step-Wise or continuous addition of an aqueous mediumof high pH value. Usually, when using moulds or yeasts and in particularwhen using Candida lipolytica, the pH of the nutrient medium will bemaintained in the range 3-6 and preferably in the range 4-5. (Bacteriarequire a higher pH usually 6.5-8.) Suitable alkaline materials foraddition to the growth mixture include sodium hydroxide, potassiumhydroxide, disodium hydrogen phosphateand am: nionia, either free or inaqueous solution.

The optimum temperature of the growth mixture will vary according to thetype of micro-organism employed and will usually lie in the range 2535C. When using Candida lipolytica the preferred temperature range is 28-32 C.

The take-up of oxygen is essential for the growth of the micro-organism.The oxygen will usually be provided as air. In order to maintain a rapidrate of growth the air, used to provide oxygen, should be present in theform of fine bubbles under the action of stirring. The air may beintroduced through a sintered surface. However there may be used thesystem of intimate aeration known as vortex aeration.

It has been found that by the use of yeast of the strain Candidalipolytica in a process according to the invention in which aeration iseffected by vortex aeration, a high growth rate is achieved whereby thegeneration time lies in the range 2-5 hours and the cell concentrationis increased by a factor of up to 12 in two days.

The micro-organism is preferably separated from the bulk of the liquidphase when possible by centrifuging and may be recovered as a cream orpaste. However, in some cases separation will be accomplished byfiltration or to some extent by decantation.

This cream or paste which contains aqueous material may be treated bycontinuous solvent extraction or by successive washings with solventfollowed by phase separation. Suitably the extraction is carried out ina stationary vessel equipped with paddle stirrer, preferably rotating atless than 10 revs per minute or in a vessel which rotates on ahorizontal axis. When operating a continuous solvent extraction, theextract is withdrawn continuously and distilled, continuously orbatchwise, at atmospheric or reduced pressure, and solvent continuouslyfed back to the extractor. troduced and withdrawn continuously orbatchwise.

Preferably the solvent extraction is effected while feeding solvent tothe extractor at a periodically varying rate to create pulsations in theflow of said liquid stream.

The pulses of the liquid passing through the solid material bring aboutoscillations and limited displacements of each grain of solid materialin relation to its neighbours, and this is equivalent to a mechanicalagitation of the whole. For this reason the whole of the products to beextracted is much more rapid and complete.

- Suitably there is arranged in the feed of the liquid stream a devicewhich imparts to it pulses whose amplitude and frequency are regulatedexperimentally at the most favourable value for each particular case.These pulses are produced by any suitable processes already known, andpreferably an alternating pump is used whose valves have been removed.

Preferably the number of pulses lies between 1 and 60 per minute. Theoperation of the process under the action of pulses is further describedin British patent application 2,234/ 63.

Suitable solvents for use in the process have been describedhereinbefore. If desired a first extraction stage can be operated usinga polar solvent, for example an alcoholic solvent, for example ethanolor isopropanol and then the partially purified micro-organism can befurther treated in a second extraction stage using a hydrocarbonsolvent, for example normal hexane or a light platformate fraction orbenzene.

Preferably in the second stage there is used as solvent a mixture. ofhydrocarbon in major amount with a polar solvent in minor amount.Preferably there is used the azeotropic mixture of hexane withisopropanol or ethanol. If desired both extraction stages can beoperated in continuous manner.

When using a solvent consisting of a mixture of hydrocarbon and a polarsolvent, it is believed that the function or one function of the polarsolvent is to weaken the bonding of the material to be extracted (eventhe. bonding of hydrocarbons which are not themselves soluble. in thepolar solvent).

Under these conditions the yeast may be in- By the use of any alcohol inthe first stage of a two stage extraction as hereinbefore described, thewater content of the micro-organism cream or paste is considerablyreduced. As a result the micro-organism containing material which is fedto the second stage has a sufficiently low content of water to ensurethat the non-aqueous contaminants which are still present are misciblewith the solevnt used in the second extraction stage. Each extractionstage may consist of either one or more substages consisting of washingwith the solvent used in the stage followed by separation.

If a single washing is employed in the first stage the amount of ethanolor isopropanol which is used should be 1.5-3 times the volume of waterwhich is present in the cream or paste of the micro-organism. However ifdesired two washings with ethanol or isopropanol may be employed usingin the first washing a volume of solvent equal to the volume of water inthe cream or paste and in the second washing a smaller amount of ethanolor isopropanol for example, one half of the amount used in the firstwashing.

Between washings of each stage or sub-stage the cream or paste isallowed to drain, for example by filtering and part of the residualsolvent is then preferably removed by vacuum filtration.

In the second stage the amount of solvent used in the (or each) washingwill usually be 2-20 times the volume of the resulting drymicro-organism.

Preferably the final stage employed for the removal of solvent isevaporation, suitably under reduced pressure and suitably in a stream ofinert gas, for example, nitrogen or super-heated steam.

By the use in the second extraction stage of a solvent which is amixture of hydrocarbon and a polar solvent the composition of the secondstage solvent, which will in any case acquire polar solvent from thefirst stage ex-' traction, can be stabilised. Build up of polar solventcan be avoided in the course of a distillation stage, in which thesecond stage solvent is recovered by the removal of separate streamsconsisting of (a) polar solvent for recycle to the first extractionstage and (b) a mixture of hydrocarbon and polar solvent for recycle tothe second stage. Suitably in a distillation stage the extract obtainedby the second extraction stage is distilled to recover (a) overhead amixture of hydrocarbon, polar solvent and water for recycle to thesecond extraction stage and (b) a bottoms fraction containing polarsolvent, water and the extracted materials; this fraciton is preferablyblended with the extract obtained in the first extraction stage beforethis is fed to distillation whereby all contaminants recovered bysolvent extraction are removed as a bottoms fraction in thisdistillation stage. Suitably the polar solvent is ethanol orisopropanol. Suitably the second stage solvent is an azeotropic mixture.

Optimum contact time will usually vary inversely with the temperature ofextraction. It will usually be undesirable to use a temperature above 70C. since higher temperatures will lead to some degradation of theproduct.

If the cream or paste of micro-organism is subjected to partial dryingbefore solvent extraction it will then usually be possible to operatethe first extraction stage with only a single washing with an alcoholicsolvent and with the use of a smaller amount of solvent than would berequired if no drying had taken place. If the extent of drying isconsiderable a first extraction stage using a polar solvent, for examplean alcoholic solvent, is not necessary; in this case the single stageextraction process can be operated by the use of a solvent which isentirely hydrocarbon or which is a mixture of hydrocarbon and polarsolvent, for example an alcohol or ketone or chlorinated hydrocarbon.

In general it is desirable to avoid drying under drastic conditionssince this will lead to partial decomposition of the micro-organism, forexample by destruction of vitamins and oxidation of unsaturatedcompounds; furthermore the products of decomposition will be soluble inthe solvent used in extractive distillation thus being lost from theproduct or requiring further stages for their recovery.

A yeast which has been freed from the whole or part of its lipids andthe contaminating hydrocarbons by one of the methods describedherinbefore and whose taste has been improved is a new industrialproduct of value for human nutrition.

The lipid extract which has been recovered by the evaporation of thesolvent is also a new industrial product which can be used either assuch or as a raw material for the separation of its sterols, fatty acids(either before saponification or after) or of its other constituents.

The invention is illustrated but not limited by the following Examples 1and 2. Experiments 1 and 2 which do not constitute operation accordingto the invention are provided for purposes of comparison.

EXAMPLE 1 A yeast cream of the strain Candida lipolytica was prepared asdescribed in copending US. application S.N. 243,961 with reference tothe diagram which accompanied said specification. It will be understoodthat the material used in the present example is the yeast creamobtained after washing with surface active yeast and with water andprior to the drying stage described with reference to said diagram.

In a series of runs a solvent was pumped continuously into an extractorcontaining a batch of the yeast cream. The extractor was in the form ofa filtration drum which was rotated with its axis horizontal.

Yeast cream containing 2 parts by Weight of dry yeast and 8 parts byweight of water was charged to the extractor with 16 parts of ethanol.The mixture was maintained at 60 C. for 30 minutes while rotating thedrum.

Solvent was drawn off, finally under vacuum. There was thus obtained amixture of 2 parts of yeast and 2 parts of solvent which consisted of /3water and /3 of ethanol, together with some remaining contaminants.

This mixture was treated with 10 parts by weight of a solvent mixtureconsisting of:

Percent by wt. Normal hexane 8O Ethanol 20 TABLE Yeast cream Yeastproduct;

Nitrogen, by wt. of dry yeast 77 7. 87 Total wt. of Lipids based on wt.of dry 0 a yeast 10% 0.5%

EXAMPLE 2 The yeast cream described in Example 1 was continuouslyextracted in a drum having its axis vertical and having a paddle stirrerwhich rotated at 10 revs/minute. In separate runs the solvent wasrespectively ethanol; ethanol followed by normal hexane; andisopropanol.

The extract was continuously distilled for the recovery of lipids andhydrocarbons and the solvent recycled to the extractor.

Conditions and results obtained were as shown in the following table:

solvent used in the first-mentioned solvent extraction is an alcohol.

TABLE Run No 4 7 g Solvent and Period of Ethanol (11 hours) Ethanolhours) Isopropanol hours) treatment. Hexane (9 hours) Rate of Feed of600 ccJhr. 600 ccJhr. 600 cc./hr.

Solvent.

Temperature C. 60 C. 60 0.

Weight of Yeast 1,000 1,000 1,000

Paste (grams).

Analysis Before After Before After Before After Treatment TreatmentTreatment Treatment Treatment Treatment Dry Yeast:

Wt. (grams 210 140 210 200 210 200 Percent N itrogem 6. 05 9. O 5. 7. 455. 65 8. 50 Total percent wt.

of Lipids in Yeast. 41. 2 5.0 42. 9 4. 2 42. 9 6. 9

Extract, Wt. (grams) 72 200 100 By Way of comparison then is providedthe following description of Experiments 1 and 2 which do not constituteoperation according to the invention.

Experiment 1 The process described in Example 1 was repeated except thatthe yeast employed was a yeast Saccharomyces cerevisial cultivated inthe known manner on a substratum of beet molasses. It is found that noperceptible improvement in the characteristic taste of this yeast isobtained.

Experiment 2 A similar experiment is carried out starting off from ayeast Torula cultivated in a known manner on cellulose bisulphiteliquor. It is found in like manner that no perceptible improvement inthe characteristic taste of this yeast is obtained.-

We claim:

1. A process which comprises cultivating a straight chainhydrocarbon-consuming micro-organism selected from the group consistingof yeasts, bacteria of the orders Pseudomonadales, Eubacteriales andActinomycetales; and Penicillium expansum in the presence of a feedstockconsisting at least in part of a straight chain hydrocarbon, separatingfrom the treated feedstock a fraction comprising the micro-organism andaqueous medium, reducing the proportion of water in said fraction bysolvent extraction using a polar solvent and thereafter subjecting theraifinate, containing the micro-organism and a reduced proportion ofaqueous medium so obtained to solvent extraction with a solventcontaining in major amount a hydrocarbon the aforementioned solventextraction.

treatment of the rafiinate being a purification step to reduceundesirable contaminants present therein.

2. A process according to claim 1 in which the straight chain paraffinichydrocarbon-consuming micro-organism is a yeast.

3. A process according to claim 2 in which the yeast is of the familyCryptococcaceae- 4. A process according to claim '3 in which the yeastis of the sub-family Cryptococcoideac. Y

5. A process according to claim 4 in which the yeast is of the genusTorulopsis.

6. A process according to claim 4 in which the yeast is of the genusCandida.

7. A process according to claim 6 in which the yeast is Candidalipolytica.

8. A process according to claim 1 in which the polar.

9. A process according to claim 8. in which the polar solvent used inthe first-mentioned solvent extraction is ethanol.

10. A process according to claim 8 in which the polar solvent used inthe first-mentioned solvent extraction is isopropanol.

11. A process according to claim 1 in which the solvent used in thesecond-mentioned solvent extraction is a mixture of a hydrocarbon and apolar solvent.

12. A process according to claim 11 in which the polar solvent used inthe second-mentioned solvent extraction is an alcohol.

13. A process according to claim 12 in which the polar solvent used inthe second-mentioned solvent extraction is ethanol.

14. A process according to claim 12 in which the polar solvent used inthe second-mentioned solvent extraction is isopropanol.

15. A process according to claim 11 in which the hydrocarbon used in thesecond-mentioned solvent extraction is normal hexane.

16. A process according to claim 11 in which the hydrocarbon used in thesecond-mentioned solvent extraction is benzene.

17. A process according to claim 11 in which the hydrocarbon used in thesecond-mentioned solvent extraction is a light platformate fraction.

18. A process according to claim 11 in which the mixed hydrocarbon andpolar solvent used in the second mentioned solvent extraction is anazeotropic mixture.

References Cited by the Examiner OTHER REFERENCES Cook, The Chemistryand Biology of Yeasts, Academic Press Inc., New York, 1958, pages648-659.

I Wickerham et al., Carbon Assimilation Tests for the Classification ofYeasts, Journal of Bacteriology, 56, 1948, pages 363-371.

A. LOUIS MONACELL, Primary Examiner. D. M. STEPHENS, Assistant Examiner.

1. A PROCESS WHICH COMPRISES CULTIVATING A STRAIGHT CHAINHYDROCARBON-CONSUMING MICRO-ORGANISM SELECTED FROM THE GROUP CONSISTINGOF YEASTS, BACTERIA OF THE ORDERS PSEUDOMONADALES, EUBACTERIALS ANDACTINOMYCETALES; AND PENCILLIUM EXPANSUM IN THE PRESENCE OF A FEEDSTOCKCONSISTING AT LEAST IN PART OF A STRAIGHT CHAIN HYDROCARBON, SEPARATINGFROM THE TREATED FEEDSTOCK A FRACTION COMPRISING THE MICRO-ORGANISM ANDAQUEOUS MEDIUM, REDUCING THE PROPORTION OF WATER IN SAID FRACTION BYSOLVENT EXTRACTION USING A POLAR SOLVENT AND THEREAFTER SUBJECTING THERAFFINATE, CONTAINING THE MICRO-ORGANISM AND A REDUCED PROPORTION OFAQUEOUS MEDIUM SO OBTAINED TO SOLVENT EXTRACTION WITH A SOLVENTCONTAINING IN MAJOR AMOUNT A HYDROCARBON THE AFOREMENTIONED SOLVENTEXTRACTION TREATMENT OF THE RAFFINATE BEING A PURIFICATION STEP TOREDUCE UNDESIRABLE CONTAIMIANT PRESENT THEREIN.